U.S. patent application number 11/916832 was filed with the patent office on 2009-03-05 for compositions and methods for treating immune disorders.
This patent application is currently assigned to Medical College of Georgia Research Institute. Invention is credited to Stephen D. Hsu, Carol A. Lapp, George S. Schuster.
Application Number | 20090062379 11/916832 |
Document ID | / |
Family ID | 37532842 |
Filed Date | 2009-03-05 |
United States Patent
Application |
20090062379 |
Kind Code |
A1 |
Hsu; Stephen D. ; et
al. |
March 5, 2009 |
COMPOSITIONS AND METHODS FOR TREATING IMMUNE DISORDERS
Abstract
Green tea polyphenol compositions and methods of their use are
provided. Certain aspects provide methods for modulating expression
of one or more autoantigens using the disclosed green tea
polyphenol compositions. Representative green tea polyphenols
include, but are not limited to (-)-epigallocatechin-3-gallate.
Other aspects provide methods for treating autoimmune disease.
Inventors: |
Hsu; Stephen D.; (Evans,
GA) ; Lapp; Carol A.; (Augusta, GA) ;
Schuster; George S.; (Augusta, GA) |
Correspondence
Address: |
Pabst Patent Group LLP
1545 PEACHTREE STREET NE, SUITE 320
ATLANTA
GA
30309
US
|
Assignee: |
Medical College of Georgia Research
Institute
|
Family ID: |
37532842 |
Appl. No.: |
11/916832 |
Filed: |
June 9, 2006 |
PCT Filed: |
June 9, 2006 |
PCT NO: |
PCT/US06/22554 |
371 Date: |
May 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60689747 |
Jun 10, 2005 |
|
|
|
Current U.S.
Class: |
514/456 |
Current CPC
Class: |
A61P 19/02 20180101;
A61K 31/7048 20130101; A61K 36/82 20130101; A61P 3/10 20180101;
A61P 7/06 20180101; A61K 31/353 20130101; A61P 37/02 20180101; A61P
17/00 20180101 |
Class at
Publication: |
514/456 |
International
Class: |
A61K 31/352 20060101
A61K031/352; A61P 37/02 20060101 A61P037/02 |
Goverment Interests
STATEMENT REGARDING FEDERAL FUNDING OR SUPPORT
[0002] Aspects of the disclosure were funded by Grant No.
R21CA097258-01 Al awarded by the National Institutes of Health. The
U.S. government may have certain rights in the claimed subject
matter.
Claims
1. A method for decreasing autoantigen expression in a cell
comprising: contacting the cell with a composition comprising one
or more green tea polyphenols, wherein the cell contacted with the
one or more green tea polyphenols has a decreased level of
autoantigen expression relative to a control.
2. The method of claim 1, wherein the one or more green tea
polyphenols comprise (-)-epigallocatechin-3-gallate or
pharmaceutically acceptable salt or prodrug thereof.
3. The method of claim 1, wherein the cell is a primary epidermal
keratinocyte, a salivary gland cell, or a lacrimal gland cell.
4. The method of claim 1, wherein the autoantigen is selected from
the group consisting of SS-A, SS-B, fodrin, centromere protein,
golgin-67, coilin, and PARP.
5. The method of claim 1, wherein the one or more green tea
polyphenols comprise (-)-epicatechin, (-)-epigallocatechin,
(-)-epicatechin-3-gallate, proanthocyanidins, enantiomers thereof,
isomers thereof, combinations thereof, and prodrugs thereof.
6. The method of claim 1, wherein expression of at least two
autoantigens is decreased relative to a control.
7. A method for modulating autoantigen gene expression comprising:
administering to a host one or more green tea polyphenols in an
amount effective to reduce or increase expression of an autoantigen
gene compared to a control.
8. The method of claim 7, wherein the one or more green tea
polyphenols comprise (-)-epigallocatechin-3-gallate or
pharmaceutically acceptable salt or prodrug thereof.
9. The method of claim 7, wherein decreased autoantigen expression
occurs in a primary epidermal keratinocyte, a salivary gland cell,
or a lacrimal gland cell.
10. The method of claim 7, wherein the autoantigen is selected from
the group consisting of SS-A, SS-B, fodrin, centromere protein,
golgin-67, coilin, and PARP.
11. The method of claim 7, wherein the one or more green tea
polyphenols comprise (-)-epicatechin, (-)-epigallocatechin,
epicatechin-3-gallate, proanthocyanidins, enantiomers thereof,
isomers thereof, combinations thereof, and prodrugs thereof.
12. The method of claim 7, wherein expression of at least two
autoantigens is decreased relative to a control.
13. A method for treating xerostomia comprising: administering to a
host one or more green tea polyphenols in an amount effective to
decrease expression of one or more autoantigens in one or more
salivary gland cells of the host.
14. A method for treating xerophthalmia comprising: administering
to a host one or more green tea polyphenols in an amount effective
to decrease expression of one or more autoantigens in one or more
of the host's lacrimal gland cells.
15. A method for treating psoriasis comprising: administering to a
host one or more green tea polyphenols in an amount effective to
decrease expression of one or more autoantigens in one or more
epidermal cells of the host.
16. The method of claim 13, wherein the one or more green tea
polyphenols comprise (-)-epigallocatechin-3-gallate or
pharmaceutically acceptable salt or prodrug thereof.
17. The method of claim 13, wherein the cell is a salivary gland
cell.
18. The method of claim 14, wherein the cell is a lacrimal gland
cell.
19. The method of claim 15, wherein the cell is a primary epidermal
keratinocyte.
20. The method of any one of claim 13, wherein the autoantigen is
selected from the group consisting of SS-A, SS-B, fodrin,
centromere protein, golgin-67, coilin, and PARP.
21. The method of claim 13, wherein the one or more green tea
polyphenols comprise (--)-epicatechin, (--)-epigallocatechin,
(--)-epicatechin-3-gallate, proanthocyanidins, enantiomers thereof,
isomers thereof, combinations thereof, and prodrugs thereof.
22. The method of claim 13, wherein expression of a least two
autoantigens is decreased relative to a control.
23. A composition comprising: an amount of
(-)-epigallocatechin-3-gallate, a pharmaceutically acceptable salt
or prodrug thereof effective to inhibit expression of one or more
autoantigens.
24. The composition of claim 23, wherein the amount of
(-)-epigallocatechin-3-gallate is effective to inhibit expression
of one or more autoantigens in a primary epidermal keratinocyte, a
salivary gland cell, or a lacrimal gland cell.
25. The composition of claim 23, wherein the autoantigen is
selected from the group consisting of SS-A, SS-B, fodrin,
centromere protein, golgin-67, coilin, and PARP.
26. The composition of claim 23, wherein the composition further
comprises (-)-epicatechin, (-)-epigallocatechin,
(-)-epicatechin-3-gallate, proanthocyanidins, enantiomers thereof,
isomers thereof, combinations thereof, or prodrugs thereof.
27. The composition of claim 23, wherein the amount of
(-)-epigallocatechin-3-gallate is effective to decrease expression
of at least two autoantigens relative to a control.
28. Use of a green tea polyphenol in the manufacture of a
medicament for the treatment of psoriasis, xerophthalmia, or
xerophthalmia.
29. Use according to claim 28, wherein the medicament comprises an
amount of green tea polyphenol effective to reduce the expression
of one or more autoantigens.
30. Use according to claim 28, wherein the one or more green tea
polyphenols comprise (-)-epigallocatechin-3-gallate or
pharmaceutically acceptable salt or prodrug thereof.
31. Use according to claim 29, wherein expression of one or more
autoantigens is decreased is a primary epidermal keratinocyte, a
salivary gland cell, or a lacrimal gland cell relative to a
control.
32. Use according to claim 29, wherein the autoantigen is selected
from the group consisting of SS-A, SS-B, fodrin, centromere
protein, golgin-67, coilin, and PARP.
33. Use according to claim 28, wherein the one or more green tea
polyphenols comprise (-)-epicatechin, (-)-epigallocatechin,
(-)-epicatechin-3-gallate, proanthocyanidins, enantiomers thereof,
isomers thereof, combinations thereof, and prodrugs thereof.
34. Use according to claim 29, wherein expression of at least two
autoantigens is decreased relative to a control.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a filing under 35 U.S.C. .sctn. 371 of
PCT/US2006/022554 filed with the U.S. Receiving Office of the
Patent Cooperation Treaty on Jun. 9, 2006, which claims benefit of
and priority to U.S. Provisional Patent Application No. 60/689,747,
filed on Jun. 10, 2005, and where permissible is incorporated by
reference in its entirety.
BACKGROUND
[0003] 1. Technical Field
[0004] Aspects of the disclosure are generally related to
compositions and methods for modulating autoantigen expression,
more particularly to green tea polyphenol compositions and methods
of their use, for example in the treatment or prophylaxis of
autoimmune disorders.
[0005] 2. Related Art
[0006] The prevalence of autoimmune disorders in the United States
is estimate at more than 8.5 million. Autoimmune reactions can
cause inflammation and apoptosis of target cells, leading to
destruction of multiple tissues and organs. Patients with
autoimmune diseases develop autoantibodies against a diverse group
of macromolecules involved in normal functions. For example,
anti-SS-A/Ro and anti-SS-B/La autoantibodies are primary markers
for certain autoimmune diseases such as lupus erythematosus and
Sjogren's syndrome. Existing treatments of autoimmune diseases have
focused on the immune system, not the autoantigens that could
trigger or sustain a positive feedback loop of inflammation and
apoptosis.
[0007] Lupus Autoimmune Disorders
[0008] Lupus is one of more than 60 types of autoimmune disorders,
and is one of the most destructive. Clinical classification of
lupus includes systematic lupus erythematosus (SLE), discoid lupus
ecrythematosus (DLE), subacute cutaneous lupus (SCLE), drug induced
lupus, and neonatal lupus. SLE is the most prevalent form and may
affect, multiple tissues such as joints, skin, kidneys, heart,
lungs, blood vessels, and brain. It affects mostly young females of
childbearing age. Lupus can cause severe joint and muscle pain,
extreme exhaustion, fevers, and skin rashes, and can lead to organ
failure, scars and death. The skin manifestations of S LE arid DLE
are slightly different. DLE affects mainly the skin and the oral
cavity with disk-shaped lesions while SLE affects multiple organs.
Skin manifestations occur in about 25% of SLE patients, with
butterfly shaped lesions distributed on the face and ears. It is
believed that cutaneous LE affects 14.6 to 68 per 100 000 people
(Callen, J. P. (2004) Br J. Dematol. 151(4):731-6).
[0009] Sjogren's Syndrome
[0010] Sjogren's syndrome (SS) is another autoimmune disorder that
affects multiple tissues. Primary SS is associated with lymphocytic
infiltrations of the salivary and lacrimal glands and eventual
atrophy, leading to a loss of fluid production. The salivary
component of SS is defined as xerostomia, with symptoms generally
referred to as salivary hypofunction (Daniels, T. E. and Fox, R. I.
(1992). Rheum Dis Clin North Am. 18(3):571-89). If not treated,
xerostomia may lead to oral complications (Daniels T. E. and Wu, A
J. (2000) Calif Dent Assoc. 28(12):933-41). Estimates of the
prevalence of SS are affected by the criteria used for diagnosis.
However, genuine differences between various regions and
communities exist (Fox, R. I. (1997) Clin Lab Med. 17(3):43 1-44;
Vitali, C. et al, (2002) Ann Rheum Dis. 61(6):554-8). The
world-wide distribution is believed to be 1/2500 (Kang, H. (1993)).
In the United States, SS affects approximately 1% of the population
(Carsons, S. (2001) Am J Manag Care. 7(14 Suppl):S433-43.24). In
China, one regional study with 26,000 subjects suggested the
prevalence of primary SS was only 0.03% (Zhang, N. (1995) Chin Med
J (Engl). 108(10):787-8). In Japan, the estimated crude prevalence
rates for SS were only 1.9 and 25.6 per 100,000 population in males
and females, respectively (Yoshida, S. (1999) Nippon Rinsho.
57(2):360-3). A survey conducted by the Japanese Ministry of Health
and Welfare indicated the SS prevalence was just 0.06% among
females (Miyasaka, N. (1995) Nippon Rinsho. 53(10):2367-70).
[0011] As for xerostomia, one study showed that among a group of
1003 Japanese individuals with an average age of 66, about 9.1%
experienced dry mouth during eating (Ikebe, K. et al. (2001) Spec
Care Dentist. 21(2):52-9), whereas in the United States, one
epidemiological study found that in a group of 2481 individuals
aged 65-84 years old, 27% reported either dry mouth or dry eyes
(Schein, O. D. et al. (1999) Arch Intern Med. 159(12): 1359-63),
and another found that dry mouth ranged from 10% among persons over
age 50 to 40% for persons over age 65 (Billings R J., et al. (1996)
Community Dent Oral Epidemiol. (5):3 12-6). Although precise
statistical comparison between the U.S. population and either the
Japanese or Chinese population is not available, it is apparent
that SS and xerostomia are more prevalent in the U.S. population,
particularly amongst the elderly.
[0012] SS is not a curable or preventable disease at present, and
whether it can be prevented or delayed is unknown. Treatment is
generally symptomatic and supportive. For xerostomia and
xerophthalmia, artificial lubricants are commonly used as saliva or
tear substitutes (Baudouin, C. et al. (2004) Rev Med Interne.
25(5):376-82). In recent years, salivary stimulants, such as
pilocarpine and cevimeline, have been approved by the FDA to treat
xerostomia (Fox, R. I. (2003) Expert Opin Investing Drugs.
12(2):247-54); Cassolato, S. F. and Turnbull, R. S. (2003)
Cerodontology. 20(2):64-77; Porter, S. R. et al. (2004) Oral Surg
Oral Med Oral Pathol Oral Radiol Endod. 97(1):28-46). In addition,
oral administration of interferon .gamma. (IFN-.gamma.) was
effective in improving saliva production in patients with primary
SS (Khurshudian, A. V. (2003) Oral Surg Oral Med Oral Pathol Oral
Radiol Endod. 95(1):38-44). However, long-term adverse effects have
not been evaluated for these therapies.
[0013] Thus, there is a need for additional compositions and
methods for preventing and treating autoimmune diseases or symptoms
associated with such diseases.
SUMMARY
[0014] Aspects of the disclosure generally provide green tea
polyphenol compositions and methods of their use, for example in
decreasing autoantigen expression in a host or cell. In particular,
it has been discovered that (-)-epigallocatechin-3-gallate
modulates expression of autoantigens. Downregulation of
autoantigens using the disclosed green tea polyphenol compositions
can be used to treat autoimmune diseases or symptoms associated
with autoimmune diseases. Increasing expression of autoantigens can
be used to assist in the purification and isolation of
autoantigens, for example to generate antibodies that can be used
as diagnostics.
[0015] One aspect of the disclosure provides a method for
decreasing autoantigen expression in a cell by contacting the cell
with a composition having one or more green tea polyphenols. The
cell contacted with the one or more green tea polyphenols shows a
decreased level of autoantigen expression relative to a control. In
certain aspects, the green tea polyphenol is
(-)-epigallocatechin-3-gallate, a pharmaceutically acceptable salt
or prodrug thereof. Representative autoantigens include those
listed in Table 1, for example SS-A, SS-B, fodrin, centromere
protein, golgin-67, coilin, and PARP. In other aspects, the
composition further includes one or more green tea polyphenols such
as (-)-epicatechin, (-)-epigallocatechin,
(-)-epicatechin-3-gallate, proanthocyanidins, enantiomers thereof
epimers thereof isomers thereof, combinations thereof, and prodrugs
thereof. It will be appreciated that the disclosed methods and
compositions can be used to modulate, in particular reduce the
expression of at least two autoantigens relative to a control.
[0016] Another aspect provides a method for modulating autoantigen
gene expression by administering to a host one or more green tea
polyphenols in an amount effective to reduce or increase expression
of an autoantigen gene compared to a control. Reduced expression of
the autoantigen gene can occur at the transcriptional or
translational level.
[0017] Still another aspect provides a method for treating an
autoimmune disease by administering to a host one or more green tea
polyphenols in an amount effective to decrease expression of one or
more autoantigens. Representative autoimmune diseases include, but
are not limited to Hashimoto's thyroiditis, pernicious anemia,
Addison's disease, type I diabetes, rheumatoid arthritis, systemic
lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus
erythematosus, multiple sclerosis, myasthenia gravis, Reiter's
syndrome, Grave's disease, scieroderma, psoriasis, xerostomia, and
xeropthalmia.
[0018] Another aspect provides a method for treating xerostomia by
administering to a host one or more green tea polyphenols in an
amount effective to decrease expression of one or more
autoantigens, wherein the decreased expression of the one or more
autoantigens occurs in one or more salivary gland cells of the
host.
[0019] Yet another aspect provides a method for treating
xerophthalmia by administering to a host one or more green tea
polyphenols in an amount effective to decrease expression of one or
more autoantigens in one or more of the host's lacrimal gland
cells.
[0020] Still another aspect provides a method for treating
psoriasis by administering to a host one or more green tea
polyphenols in an amount effective to decrease expression of one or
more autoantigens in one or more epidermal cells of the host.
[0021] Another aspect provides a use of a green tea polyphenol in
the manufacture of a medicament for the treatment of an autoimmune
disease, in particular, psoriasis, xerostomia, or
xerophthalmia.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows a gel indicating mRNA levels of SS-B/La and
SS-A/Ro in normal, immortalized, and tumor cells treated with
(-)-epigallocatechin-3-gallate.
[0023] FIG. 2 shows Western blot results of autoantigen protein
levels in (-)-epigallocatechin-3-gallate-treated NHEK and NS-SV-Ac
cells.
[0024] FIG. 3 shows mice treated with green tea polyphenols have
reduced autoantibodies.
[0025] FIG. 4A shows submandibular gland sections from control NOD
mice.
[0026] FIG. 4B shows submandibular gland sections from NOD mice fed
with green tea polyphenols.
[0027] FIG. 5 shows a bar graph of the densities of the average
focal areas in NOD mice fed green tea polyphenols compared to
control mice.
[0028] FIG. 6 shows a bar graph of cell viability indicating that
ECGC protects cells against TNF-induced cytotoxicity.
[0029] FIG. 7 A shows a bar graph of cell viability indicating that
inhibition of p38 abolishes EGCG protection.
[0030] FIG. 7B shows a bar graph of cell viability indicating that
inhibition of MEK abolishes the EGCG effect.
[0031] FIG. 8 shows an autoradiograph indicating that p38 is
rapidly and specifically phosphorylated within 30 min in acinar
cell-derived NS-SV-AC cells contacted with EGCG.
[0032] FIG. 9 shows micrographs indicating local lymphocyte
infiltration in the submandibular glands of early GTP-treated MRL
and NOD mice.
DETAILED DESCRIPTION
1. Definitions
[0033] Before explaining the various embodiments of the disclosure,
it is to be understood that the invention is not limited in its
application to the details of construction and the arrangement of
the components set forth in the following description. Other
embodiments can be practiced or carried out in various ways. Also,
it is to be understood that the phraseology and terminology
employed herein is for the purpose of description and should not be
regarded as limiting.
[0034] Throughout this disclosure, various publications, patents
and published patent specifications are referenced. Where
permissible, the disclosures of these publications, patents and
published patent specifications are hereby incorporated by
reference in their entirety into the present disclosure to more
fully describe the state of the art.
[0035] To facilitate understanding of the disclosure, the following
definitions are provided:
[0036] As used herein, the singular forms "a," "an," and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "a factor" refers to one
or mixtures of factors, and reference to "the method of treatment"
includes reference to equivalent steps and methods known to those
skilled in the art, and so forth.
[0037] The term "autoantigen" refers to an antigen produced by an
organism and recognized by the organism's immune system.
Representative autoantigens include, but are not limited to those
listed in Table 1.
[0038] The term "autoimmune disease or disorder" refers to
conditions caused by an immune response against the body's own
tissues or cells. Representative autoimmune disorders include, but
are not limited to Hashimoto's thyroiditis, pernicious anemia,
Addison's disease, type I diabetes, rheumatoid arthritis, systemic
lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus
erythematosus, multiple sclerosis, myasthenia gravis, Reiter's
syndrome, Grave's disease, scleroderma, psoriasis, xerostomia, and
xeropthalmia.
[0039] The term "cell" refers to a membrane-bound biological unit
capable of replication or division.
[0040] The term "Green Tea Polyphenols or GTP" refers to
polyphenolic compounds present in the leaves of Carmellia sinensis.
Green tea polyphenols include, but are not limited to
(-)-epicatechin (EC), (-)-epigallocatechin (EGC),
(-)-epicatechin-3-gallate (ECF), (-)-epigallocatechin-3-gallate
(ECGC), proanthocyanidins, enantiomers thereof, epimers thereof
isomers thereof, combinations thereof, and prodrugs thereof.
[0041] The term "host" refers to a living organism, including but
not limited to a mammal such as a primate, and in particular a
human.
[0042] The term "isolated," when used to describe the various
compositions disclosed herein, means a substance that has been
identified and separated and/or recovered from a component of its
natural environment. For example an isolated polypeptide or
polynucleotide is free of association with at least one component
with which it is naturally associated. Contaminant components of
its natural environment are materials that would typically
interfere with diagnostic or therapeutic uses for the polypeptide
or polynucleotide and may include enzymes, and other proteinaceous
or non-proteinaceous solutes. An isolated substance includes the
substance in situ within recombinant cells. Ordinarily, however, an
isolated substance will be prepared by at least one purification
step.
[0043] The term "operably linked" refers to a juxtaposition wherein
the components are configured so as to perform their usual
function. For example, control sequences or promoters operably
linked to a coding sequence are capable of effecting the expression
of the coding sequence, and an organelle localization sequence
operably linked to protein will direct the linked protein to be
localized at the specific organelle.
[0044] The term "pharmaceutically" acceptable carrier" refers to a
carrier or diluent that does not cause significant irritation to an
organism and does not abrogate the biological activity and
properties of the administered compound.
[0045] The term "pharmaceutically acceptable salt" refers to those
salts which retain the biological effectiveness and properties of
the free bases and which are obtained by reaction with inorganic or
organic acids such as hydrochloric acid, hydrobromic acid, sulfuric
acid, nitric acid, phosphoric acid, methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic
acid, maleic acid, succinic acid, tartaric acid, citric acid, and
the like.
[0046] A "pharmaceutical composition" refers to a mixture of one or
more of the green tea polyphenols described herein, or a
pharmaceutically acceptable salts thereof, with other chemical
components, such as physiologically acceptable carriers and
excipients. The purpose of a pharmaceutical composition is to
facilitate administration of a compound to an organism.
[0047] The term "prodrug" refers to an agent, including nucleic
acids and proteins, which is converted into a biologically active
form in vivo. Prodrugs are often useful because, in some
situations, they may be easier to administer than the parent
compound. They may, for instance, be bioavailable by oral
administration whereas the parent compound is not. The prodrug may
also have improved solubility in pharmaceutical compositions over
the parent drug. A prodrug may be converted into the parent drug by
various mechanisms, including enzymatic processes and metabolic
hydrolysis. Harper, N. J. (1962). Drug Latentiation in Jucker, ed.
Progress in Drug Research, 4:221-294; Morozowich et al. (1977).
Application of Physical Organic Principles to Prodrug Design in E.
B. Roche ed. Design of Biopharmaceutical Properties through
Prodrugs and Analogs, APhA; Acad. Pharm, Sci.; E. B. Roche, ed.
(1977). Bioreversible Carriers in Drug in Drug Design, Theory and
Application, APhA; H. Bundgaard, ed. (1985) Design of Prodrugs,
Elsevier; Wang et al. (1999) Prodrug approaches to the improved
delivery of peptide drug, Curr. Pharm. Design. 5(4):265-287;
Pauletti et al. (1997). Improvement in peptide bioavailability:
Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev.
27:235-256; Mizen et al. (1998). The Use of Esters as Prodrugs for
Oral Delivery of [beta]-Lactam antibiotics, Pharm. Biotech.
11,:345-365; Gaignault et al. (1996). Designing Prodrugs and
Bioprecursors I. Carrier Prodrugs, Pract. Med. Chem. 671-696; M.
Asgharnejad (2000). Improving Oral Drug Transport Via Prodrugs, in
G. L. Amidon, P. I. Lee and E. M. Topp, Eds., Transport Processes
in Pharmaceutical Systems, Marcell Dekker, p. 185-218; Balant et
al. (1990) Prodrugs for the improvement of drug absorption via
different routes of administration, Eur. J. Drug Metab.
Pharmacokinet, 15(2): 143-53; Balimane and Sinko (1999).
Involvement of multiple transporters in the oral absorption of
nucleoside analogues, Adv. Drug Delivery Rev., 39(1-3): 183-209;
Browne (1997). Fosphenyloin (Cerebyx), Clin. Neuropharmacol. 20(1):
1-12; Bundgaard (1979). Bioreversible derivatization of
drugs--principle and applicability to improve the therapeutic
effects of drugs, Arch. Pharm. Chemi. 86(1): 1-39; H. Bundgaard,
ed. (1985) Design of Prodrugs, New York: Elsevier; Fleisher et al.
(1996). Improved oral drug delivery: solubility limitations
overcome by the use of prodrugs, Adv. Drug Delivery Rev. 19(2):
115-130; Fleisher et al. (1985). Design of prodrugs for improved
gastrointestinal absorption by intestinal enzyme targeting, Methods
Enzymol. 112: 360-81; Farquhar D, et al. (1983). Biologically
Reversible Phosphate-Protective Groups, J. Pharm. Sci., 72(3):
324-325; Han, H. K. et al. (2000). Targeted prodrug design to
optimize drug delivery, AAPS PharmSci., 2(1): E6; Sadzuka Y.
(2000). Effective prodrug liposome and conversion to active
metabolite, Curr Drug Metab., l(1):31-48; D. M. Lambert (2000)
Rationale and applications of lipids as prodrug carriers, Eur. J.
Pharm. Sci., 11 Suppl 2:S15-27; Wang, W. et al. (1999) Prodrug
approaches to the improved delivery of peptide drugs. Curr. Pharm.
Des., 5(4).265-87.
[0048] The term "treating or treatment" refers to alleviating,
reducing, or inhibiting one or more symptoms or physiological
aspects of a disease, disorder, syndrome, or condition.
2. Green Tea Compositions
[0049] One embodiment provides a composition having one or more
green tea polyphenols, in particular
(-)-epigallocatechin-3-gallate, a pharmaceutically acceptable salt,
prodrug, or derivative thereof, in an amount effective to modulate
expression of one or more autoantigens in a host compared to a
control. Modulate means to increase, decrease, reduce, or inhibit
expression of an autoantigen in a host or cell. Experimental
controls or control groups are known in the art. Generally, the
effect of the green tea polyphenol composition on the
downregulation, inhibition, or modulation of an autoantigen can be
compared to the effect of the composition without the green tea
polyphenol on the down regulation, inhibition, or modulation of an
autoantigen. Representative hosts include mammals such as
humans.
[0050] A derivative or variant of a green tea polyphenol includes
green tea polyphenols having chemical modifications to increase
solubility or bioavailability in a host. These chemical
modifications include the addition of chemical groups having a
charge under physiological conditions as well as the conjugation of
the green tea polyphenol to other biological moieties such as
polypeptides, carbohydrates, lipids, or a combination thereof.
[0051] The disclosed green tea polyphenol composition can decrease
or inhibit the expression of an autoantigen in any cell expressing
an autoantigen or capable of expressing an autoantigen.
Representative cells include, but are not limited to a primary
epidermal keratinocyte, a salivary gland cell, or a lacrimal gland
cell.
[0052] Autoantigens include, but are not limited to anti-nuclear
autoantibodies such as SS-A/Ro, SS-B/La, centromere protein (CNEP)
A, B, C, dsDNA, polymyositis-scleroderma (PM-scl), RNA polymerases,
poly(ADP)ribose polymerase (PARP), uridine rich 1 small nuclear
ribonucleoprotein (Ul snRNP), Smith antigen (Sm), ribosomal-P,
histidyl t-RNA synthase (Jo-1), and DNA topoisomerase 1 (Scl-70) as
well as those listed in Table 1.
[0053] Another embodiment provides a pharmaceutical composition
including one or more green tea polyphenols in combination with a
pharmaceutically acceptable carrier, diluent, or excipient. The one
or more green tea polyphenols are in an amount modulate the
expression of an autoantigen in a host. In some embodiments, the
one or more green tea polyphenols are in an amount effective to
inhibit, reduce, or decrease the expression of two or more
autoantigens in a host. In other embodiments, the active ingredient
in the composition consists essentially of
(-)-e[rho]igallocatechin-3-gallate, a pharmaceutically acceptable
salt or prodrug thereof. The active ingredient can be in the form a
single optical isomer. Typically, one optical isomer will be
present in greater than 85%, 90%, 95%, or 99% by weight compared to
the other optical isomer. It will be appreciated that the
composition can also include at least one additional active
ingredient, for example a second therapeutic. Additional
description of the disclosed pharmaceutical compositions is
provided below.
3. Methods of Use
[0054] One embodiment provides a method for modulating expression
of one or more autoantigens in a cell or host by contacting the
cell or host with a green tea polyphenol composition. The green tea
polyphenol composition includes an amount of one or more green tea
polyphenols, pharmaceutically acceptable salts, prodrugs, or
derivatives thereof in an amount effective to modulate the
expression of an autoantigen. The expression of the autoantigen can
be increased or decreased as compared to a control. The modulation
of autoantigen expression can be at the transcriptional or
translational stage. For example, the amount of mRNA encoding one
or more autoantigens can be reduced or increased in cells contacted
with a green tea polyphenol composition relative to a control.
Alternatively, the amount of protein corresponding to an
autoantigen can be increased or reduced in cells contacted with the
disclosed green tea polyphenol compositions.
[0055] Another embodiment provides a method for treating an
autoimmune disease by administering to a host an amount of one or
more green tea polyphenols effective to reduce, inhibit, or
decrease the expression of an autoantigen relative to a control.
Representative autoimmune diseases include, but are not limited to
Hahimoto's thyroiditis, pernicious anemia, Addison's disease, type
I diabetes, rheumatoid arthritis, systemic lupus erythematosus
(SLE), dermatomyositis, Sjogren's syndrome (SS), lupus
erythematosus, multiple sclerosis, myasthenia gravis, Reiter's
syndrome, Grave's disease, scleroderma, psoriasis, xerostomia, and
xeropthlmia. Both SLE and SS are characterized by the production of
autoantibodies that have been implicated in the pathogenic effects
on tissues. To date, a large number of autoantigens have been
identified in SLE. Sera from lupus patients often have high titers
of anti-nuclear autoantibodies (ANAs) that target components of the
nucleus (Sawalha and Harley, (2004) Curr Opin Rheumatol.
16(5):534-40). These ANAs include SS-A/Ro, SS-B/La, centromere
protein (CNEP) A, B, C, dsDNA, polymyositis-scleroderma (PM-scl),
RNA polymerases, [rho]oly(ADP)ribose polymerase (PARP), uridine
rich 1 small nuclear ribonucleoprotein (Ul snRNP), Smith antigen
(Sm), ribosomal-P, histidyl t-RNA synthase (Jo-1), and DNA
topoisomerase 1 (Scl-70) (Reeves, G. E. (2004) Intern Med J.
34(6):338-7). ANAs are also found in about 70% of patients with SS,
and autoantibodies against SS-A/Ro and SS-B/La are found in about
95% and 87% of primary SS patients, respectively (Rehman H. U.
(2003) Yonsei Med J. 44(6):947-54). Elevated levels of SS-A/Ro and
SS B/La mRNA were found in the salivary tissues of SS patients with
xerostomia (Bolstad A. I., et al. (2003) Arthritis Rheum
48:174-85). Lupus-associated autoantigens also include golgins
present in the Golgi apparatus and coilin proteins (Stinton, L. M.
et al. (2004) Clin Immunol. 110(1):30-44).
[0056] The mechanism leading to presentation of autoantigens to the
immune system is unknown. One mechanism that may initiate the
autoimmune response is the translocation of nuclear autoantigens
onto the cell membrane during apoptosis, where they are exposed to
antigen-presenting cells such as macrophages and dendritic cells
(Manganelli and Fietta, 2003). During apoptosis, autoantigens
redistribute to form apoptotic bodies and blebs, where autoantigens
such as SS-A/Ro, SS-B/La, Ku, poly(ADP)ribose polymerase (PARIP),
fodrin, Golgins and nuclear mitotic apparatus protein (NuMA) are
clustered as subcellular structures. An aberrant structure of these
autoantigen complexes may contribute to the autoimmune response
(Rosen and Casciola-Rosen, 2004). B cells can be stimulated to
proliferate and produce autoantibodies by perturbations in the
levels of cytokines. Although the exact role of autoantibodies in
the pathogenesis SLE or SS remains unclear, it is thought they are
involved directly in some of the clinical manifestations (Mamula et
al, 1994).
[0057] Another embodiment provides a method for treating an
autoimmune disease by administering to a host an amount of green
tea polyphenol, for example ECGC, effective to down-regulate the
expression of autoantigens at the mRNA and/or protein levels, for
example in different epithelial cell types. Still another
embodiment provides a method for treating an autoimmune disease or
disorder by administering to a host an amount of green tea
polyphenol, for example ECGC, effective to reduce or inhibit
expression of an autoantigen, reduce or inhibit apoptosis; and
reduce or inhibit inflammation. The reduction in expression of an
autoantigen, reduction of apoptosis, and the reduction of
inflammation can be in any cell capable of expressing an
autoantigen, for example in epithelial cells such as salivary gland
cells, lacrimal gland cells, or primary epithelial
keratinocyte.
[0058] Results from the Affymetrix gene expression analyses in
Example I and Table I indicated that EGCG modulated the expression
of a group of major autoantigens in NHEK, with several genes
showing a 2-fold or more reduction in mRNA levels, in some cases
after an initial increase. The various different patterns in the
kinetics of change among different autoantigens suggests that
different regulator mechanisms could be involved. (McArthur et al,
2002). Expression of SS-A/Ro 52 (which was not represented on the
Affynatrix array) was shown to be reduced by ECCG at the mRNA and
protein level in two different epithelial cell lines using RT-PCR
and Western analyses. Similarly, microarray, RT-PCR and Western
analyses demonstrated that EGCG reduced expression of SS-B/La. In
contrast, the microarray analysis showed that SS-A/Ro 60 mRNA
levels were not significantly altered by EGCG. Interestingly,
oxidative stress induces cell surface expression of SS-A/Ro 52, but
not SS-A/Ro 60 autoantigen on NHEK cells (Saegusa et al. 2002).
Since green tea polyphenols inhibit the effects of oxidative stress
on normal cells, this may be one mechanism by which EGCG reduces
expression of autoantigens (Yamamotu et al, 2004). An inhibitory
effect of EGCG on protein levels of four other autoantigens was
demonstrated by Western analysis in NHEK and NS-SV-AC cells. The
kinetics of reduction in protein levels differed somewhat between
the autoantigens. This could reflect regulation via different
pathways, or differences in mRNA or protein stability, or in
protein trafficking.
[0059] Another embodiment provides a method for reducing or
inhibiting SS-induced salivary gland destruction by administering
to a host expressing one more symptoms of SS an amount of green tea
polyphenols effective to reduce or inhibit one or more of
apoptosis, autoantigen gene expression, or cytokine production.
Examples 4 and 5 show a significant reduction of serum total
autoantibody levels in GTP-treated NOD animals, compared with the
untreated control NOD animals (FIG. 3). The NOD mouse is a known
model for SS. Similarly, the size of lymphocytic infiltrate foci
was also reduced after GTP treatment (FIGS. 4 and 5). Thus, another
embodiment provides a method for reducing lymphocytic infiltration
of salivary glands by administering to a host an amount of green
tea polyphenols, for example ECGC, effective to reduce or inhibit
the expression of an autoantigen.
[0060] The disclosed in vivo evidence indicates that GTPs have a
beneficial effect against autoimmune responses in the NOD mouse.
Green tea consumption by humans leads to an increase of secreted
salivary GTPs, in a concentration range (50+.mu.M) 10 times higher
than the serum levels (Yang et al, 1999). Oral exposure to GTPs in
this mouse model also results in elevation of salivary GTPs to
protective concentrations. The GTP-treated group also had an
average one week delay in the onset of autoimmune diabetes, and
while all of the 15 GTP-fed NOD mice survived the 3-week disease
progression period, two untreated control mice died during this
period.
[0061] Although the size of the foci showed a significant
difference between the two groups, the focal scores based on human
diagnostic criteria did not differ. This could be due to species
differences or more subtle differences between human SS and the NOD
mouse model. A further possibility is that the time of onset of GTP
treatment (9 weeks of age) might be relatively late with respect to
the initial phases of the process of gland damage. NOD-scid
congenic mice (that lack functional lymphocytes) do not develop
sialadenitis (or diabetes). However, they do show dysfunction in
expression of biochemical markers of salivary gland differentiation
such as amylase and parotid secretory protein (PSP). These data are
consistent with a model for SS in which there is an initial phase,
during which dysregulation of glandular homeostasis triggers the
disease, followed by an immune cell-mediated phase that leads to a
loss of secretory function (Cha et al, 2002).
[0062] Still another embodiment provides a method for reducing or
inhibiting autoimmune destruction of cells expressing an
autoantigen by contacting the cells with an amount of green tea
polyphenols, for example ECGC, effective to activate the p38
pathway in the cells.
[0063] The multiple MAPK signal transduction pathways are involved
in the control of diverse cellular events including proliferation,
differentiation and apoptosis. Gene expression in salivary
epithelial cells is regulated, in part, via the Raf/MEK/MAPK
pathway (Slomiany and Slomiany, 2003, Li et al, 1997). It was found
that Raf-1 kinase-induced down-regulation of a sodium channel was
blocked by the MEK inhibitor PD98059, suggesting that the ERK
pathway is involved in the signal transduction (Zentner et al,
1998). The acinar cells respond to nitric oxide (NO), an
inflammation-related signaling molecule, by the pathways regulated
by ERK and p38 (Slomiany and Slomiany, 2002a). The p38 MAPK pathway
is important in transducing stress signals, and p38 MAPK is
strongly and rapidly activated by stresses and inflammatory
cytokines (Dent et al, 2003). Recently, it was suggested that
inhibition of LP S-stimulated iNOS and COX-2 expression and reduced
NO release were by a mechanism involving p38 (Brautigam et al,
2005). SS patients show activated forms of p38 and JNK in
infiltrating mononuclear cells (Nakamura et al, 1999). Protein
kinases downstream of p38 can activate transcription factors such
as activating transcription factor-2 (ATF-2) and growth and DNA
damage (GADD)-153 transcription factor. The p38 MAPK family
consists of at least 4 isoforms. The specificity of the isoforms
activated depends on the cell type, and the nature and strength of
the signals (Morin and Huot, 2004). Importantly, the cellular
response to p38 MAPK activation is highly cell type dependent: it
can induce apoptosis, growth arrest, or differentiation (Slomiany
and Slomiany, 2002, Dent et al, 2003, Morin and Huot, 2004).
[0064] It has been discovered that EGCG induced the activation of
p38 by phosphorylation in a dose dependent manner. p38 is rapidly
and specifically phosphorylated within 30 min in acinar
cell-derived NS-SV-AC cells by EGCG, while levels pJNK and pFRK
were relatively stable (FIG. 8). Further, when NS-SV-AC cells were
pre-treated with a specific inhibitor of p38, EGCG failed to
protect these cells from TNF-.alpha.-induced cytotoxicity (FIG.
7A). An inhibitor of MEK, a MAPK upstream of p38, also blocked EGCG
protection (FIG. 7B). Taken together, these results implicate the
p38/MAPK pathway in mediation of some of the beneficial effects of
GTPs on cell-based mechanisms of disease in SS-affected salivary
glands. Accordingly, another embodiment provides a method for
treating an autoimmune disorder by administering to a host an
activator of the p38/MAPK pathway, in particular a green tea
polyphenol compound.
[0065] Still another embodiment provides a method for treating
xerostomia by administering to a host one or more green tea
polyphenols in an amount effective to decrease expression of one or
more autoantigens in one or more salivary gland cells of the host.
Reducing the expression of an autoantigen in the salivary gland
cell can reduce the autoimmune destruction of the salivary gland
cell.
[0066] Another embodiment provides a method for treating
xerophthalmia by administering to a host one or more green tea
polyphenols in an amount effective to decrease expression of one or
more autoantigens in one or more of the host's lacrimal gland
cells. Reducing the expression of an autoantigen can prevent
destruction of lacrimal gland cells.
[0067] Still another embodiment provides a method for treating
psoriasis by administering to a host one or more green tea
polyphenols in an amount effective to decrease expression of one or
more autoantigens in one or more epidermal cells of the host.
Reducing the expression of an autoantigen can prevent autoimmune
destruction of epidermal cells.
[0068] Another embodiment provides use of a green tea polyphenol in
the manufacture of a medicament for the treatment of an autoimmune
disease, for example SS, psoriasis, xerophthalmia, or
xerophthalmia.
4. Combination Therapy
[0069] The disclosed green tea polyphenol compositions can be used
in combination or alternation with one or more additional
therapies. Representative additional therapies that can be used
with the disclosed compositions include, but are not limited to
non-steroidal anti-inflammatory drugs, antimalarial drugs,
corticosteroids, immunosuppressants, antioxidants, antibodies
against T-cells or TNF-[alpha], and combinations thereof. Suitable
non-steroidal anti-inflammatory drugs include ibuprofen, naproxen,
indomethacin, celecoxib or other COX-2 inhibitors, rofecoxib, and
combinations thereof. Treatment for psoriasis includes PUVA (UV
light plus psorialin). Exemplary antimalarials include
hydroxychloroquine. Representative corticosteroids include
prednisone, prednisolone, and combinations thereof. Suitable
immunosuppressants include azathioprine, methotrexate,
cyclosporine, cyclophosphamide, leflunomide, mycophenolate, and
combinations thereof. Additionally, herb al extracts,
nutraceuticals, vitamins or combinations thereof can be used with
or in addition to the disclosed compositions.
5. Pharmaceutical Compositions
[0070] Another embodiment provides pharmaceutical compositions and
dosage forms which include a pharmaceutically acceptable salt of
one or more green tea polyphenols, in particular,
(-)-epigallocatechin-3-gallate or a pharmaceutically acceptable
polymorph, solvate, hydrate, dehydrate, co-crystal, anhydrous, or
amorphous form thereof. Specific salts of disclosed compounds
include, but are not limited to, sodium, lithium, potassium salts,
and hydrates thereof.
[0071] Pharmaceutical unit dosage forms of green tea polyphenols
are suitable for oral, mucosal (e.g., nasal, sublingual, vaginal,
buccal, or rectal), parenteral (e.g., intramuscular, subcutaneous,
intravenous, intraarterial, or bolus injection), topical, or
transdermal administration to a patient. Examples of dosage forms
include, but are not limited to: tablets; caplets; capsules, such
as hard gelatin capsules and soft elastic gelatin capsules;
cachets; troches; lozenges; dispersions; suppositories; ointments;
cataplasms (poultices); pastes; powders; dressings; creams;
plasters; solutions; patches; aerosols (e.g., nasal sprays or
inhalers); gels; liquid dosage forms suitable for oral or mucosal
administration to a patient, including suspensions (e.g., aqueous
or non-aqueous liquid suspensions, oil-in-water emulsions, or
water-in-oil liquid emulsions), solutions, and elixirs; liquid
dosage forms suitable for parenteral administration to a patient;
and sterile solids (e.g., crystalline or amorphous solids) that can
be reconstituted to provide liquid dosage forms suitable for
parenteral administration to a patient.
[0072] The composition, shape, and type of dosage forms of the
green tea polyphenols of the disclosure will typically vary
depending on their use. A parenteral dosage form may contain
smaller amounts of the active ingredient than an oral dosage form
used to treat the same disease or disorder. These and other ways in
which specific dosage forms encompassed by this disclosure will
vary from one another will be readily apparent to those skilled in
the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed.,
Mack Publishing, Easton, Pa. (1990).
[0073] Pharmaceutical compositions and unit dosage forms of the
disclosure typically also include one or more pharmaceutically
acceptable excipients or diluents. Advantages provided by specific
compounds of the disclosure, such as, but not limited to, increased
solubility and/or enhanced flow, purity, or stability (e.g.,
hygroscopicity) characteristics can make them better suited for
pharmaceutical formulation and/or administration to patients than
the prior art. Suitable excipients are well known to those skilled
in the art of pharmacy or pharmaceutics, and non-limiting examples
of suitable excipients are provided herein. Whether a particular
excipient is suitable for incorporation into a pharmaceutical
composition or dosage form depends on a variety of factors well
known in the art including, but not limited to, the way in which
the dosage form will be administered to a patient. For example,
oral dosage forms such as tablets or capsules may contain
excipients not suited for use in parenteral dosage forms. The
suitability of a particular excipient may also depend on the
specific active ingredients in the dosage form. For example, the
decomposition of some active ingredients can be accelerated by some
excipients such as lactose, or when exposed to water. Active
ingredients that include primary or secondary amines are
particularly susceptible to such accelerated decomposition.
[0074] The disclosure further encompasses pharmaceutical
compositions and dosage forms that include one or more compounds
that reduce the rate by which an active ingredient, for example a
green tea polyphenol, will decompose. Such compounds, which are
referred to herein as "stabilizers," include, but are not limited
to, antioxidants such as ascorbic acid, pH buffers, or salt
buffers. In addition, pharmaceutical compositions or dosage forms
of the disclosure may contain one or more solubility modulators,
such as sodium chloride, sodium sulfate, sodium or potassium
phosphate or organic acids. A specific solubility modulator is
tartaric acid.
[0075] Like the amounts and types of excipients, the amounts and
specific type of green tea polyphenol in a dosage form may differ
depending on factors such as, but not limited to, the route by
which it is to be administered to patients. However, typical dosage
forms of the compounds of the disclosure include a pharmaceutically
acceptable salt, or a pharmaceutically acceptable polymorph,
solvate, hydrate, dehydrate, co-crystal, anhydrous, or amorphous
form thereof, in an amount of from about 10 mg to about 1000 mg,
preferably in an amount of from about 25 mg to about 750 mg, more
preferably in an amount of from 50 mg to 500 mg, even more
preferably in an amount of from about 30 mg to about 100 mg.
[0076] Additionally, the compounds and/or compositions can be
delivered using lipid- or polymer-based nanoparticles. For example,
the nanoparticles can be designed to improve the pharmacological
and therapeutic properties of drugs administered parenterally
(Allen, T. M., Cullis, P. R. Drug delivery systems: entering the
mainstream. Science. 303(5665):1818-22 (2004)).
[0077] Topical dosage forms of the disclosure include, but are not
limited to, creams, lotions, ointments, gels, sprays, aerosols,
solutions, emulsions, and other forms know to one of skill in the
art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack
Publishing, Easton, Pa. (1990); and Introduction to Pharmaceutical
Dosage Forms, 4th ed., Lea & Febiger, Philadelphia, Pa. (1985).
For non-sprayable topical dosage forms, viscous to semi-solid or
solid forms including a carrier or one or more excipients
compatible with topical application and having a dynamic viscosity
preferably greater than water are typically employed. Suitable
formulations include, without limitation, solutions, suspensions,
emulsions, creams, ointments, powders, liniments, salves, and the
like, which are, if desired, sterilized or mixed with auxiliary
agents (e.g., preservatives, stabilizers, wetting agents, buffers,
or salts) for influencing various properties, such as, for example,
osmotic pressure. Other suitable topical dosage forms include
sprayable aerosol preparations wherein the active ingredient,
preferably in combination with a solid or liquid inert carrier, is
packaged in a mixture with a pressurized volatile (e.g., a gaseous
propellant, such as freon), or in a squeeze bottle. Moisturizers or
humectants can also be added to pharmaceutical compositions and
dosage forms if desired. Examples of such additional ingredients
are well known in the art. See, e.g., Remington's Pharmaceutical
Sciences, 18th Ed., Mack Publishing, Easton, Pa. (1990).
[0078] Transdermal and mucosal dosage forms of the compositions of
the disclosure include, but are not limited to, ophthalmic
solutions, patches, sprays, aerosols, creams, lotions,
suppositories, ointments, gels, solutions, emulsions, suspensions,
or other forms known to one of skill in the art. See, e.g.,
Remington's Pharmaceutical Sciences, 11 th Ed., Mack Publishing,
Easton, Pa. (1990); and Introduction to Pharmaceutical Dosage
Forms, 4th Ed., Lea & Febiger, Philadelphia, Pa. (1985). Dosage
forms suitable for treating mucosal tissues within the oral cavity
can be formulated as mouthwashes, as oral gels, or as buccal
patches. Additional transdermal dosage forms include "reservoir
type" or "matrix type" patches, which can be applied to the skin
and worn for a specific period of time to permit the penetration of
a desired amount of active ingredient.
[0079] Examples of transdermal dosage forms and methods of
administration that can be used to administer the green tea
polyphenols of the disclosure include, but are not limited to,
those disclosed in U.S. Pat. Nos. 4,624,665; 4,655,767; 4,687,481;
4,797,284; 4,810,499; 4,834,978; 4,877,618; 4,880,633; 4,917,895;
4,927,687; 4,956,171; 5,035,894; 5,091,186; 5,163,899; 5,232,702;
5,234,690; 5,273,755; 5,273,756; 5,308,625; 5,356,632; 5,358,715;
5,372,579; 5,421,816; 5,466;465; 5,494,680; 5,505,958; 5,554,381;
5,560,922; 5,585,111; 5,656,285; 5,667,798; 5,698,217; 5,741,511;
5,747,783; 5,770,219; 5,814,599; 5,817,332; 5,833,647; 5,879,322;
and 5,906,830, each of which are incorporated herein by reference
in their entirety.
[0080] Suitable excipients (e.g., carriers and diluents) and other
materials that can be used to provide transdermal and mucosal
dosage forms encompassed by this disclosure are well known to those
skilled in the pharmaceutical arts, and depend on the particular
tissue or organ to which a given pharmaceutical composition or
dosage form will be applied. With that fact in mind, typical
excipients include, but are not limited to, water, acetone,
ethanol, ethylene glycol, propylene glycol, butane-1,3-diol,
isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures
thereof, to form dosage forms that are non-toxic and
pharmaceutically acceptable.
[0081] Depending on the specific tissue to be treated, additional
components may be used prior to, in conjunction with, or subsequent
to treatment with pharmaceutically acceptable salts of a green tea
polyphenol of the disclosure. For example, penetration enhancers
can be used to assist in delivering the active ingredients to or
across the tissue. Suitable penetration enhancers include, but are
not limited to: acetone; various alcohols such as ethanol, oleyl,
an tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide;
dimethyl acetamide; dimethyl formamide; polyethylene glycol;
pyrrolidones such as polyvinylpyrrolidone; Kollidon grades
(Povidone, Polyvidone); urea; and various water-soluble or
insoluble sugar esters such as TWEEN 80 (polysorbate 80) and SPAN
60 (sorbitan monostearate).
[0082] The pH of a pharmaceutical composition or dosage form, or of
the tissue to which the pharmaceutical composition or dosage form
is applied, may also be adjusted to improve delivery of the active
ingredient(s). Similarly, the polarity of a solvent carrier, its
ionic strength, or tonicity can be adjusted to improve delivery.
Compounds such as stearates can also be added to pharmaceutical
compositions or dosage forms to advantageously alter the
hydrophilicity or lipophilicity of the active ingredient(s) so as
to improve delivery. In this regard, stearates can serve as a lipid
vehicle for the formulation, as an emulsifying agent or surfactant,
and as a delivery-enhancing or penetration-enhancing agent.
Different hydrates, dehydrates, co-crystals, solvates, polymorphs,
anhydrous, or amorphous forms of the pharmaceutically acceptable
salt of a tight junction modulator can be used to further adjust
the properties of the resulting composition.
[0083] The disclosed green tea polyphenol compositions can also be
formulated as extended or delayed release formulations, Extended
and delayed release formulations for various active ingredients are
known in the art, for example by encapsulation.
6. Encapsulation of Green Tea Polyphenols
[0084] In another embodiment, the green tea polyphenols can be
incorporated into a polymeric component by encapsulation in a
microcapsule. The microcapsule can be fabricated from a material
different from that of the bulk of the carrier, coating, or matrix.
Suitable microcapsules are those which are fabricated from a
material that undergoes erosion in the host or those which are
fabricated such that they allow the green tea polyphenol to diffuse
out of the microcapsule. Such microcapsules can be used to provide
for the controlled release of the encapsulated green tea polyphenol
from the microcapsules.
[0085] Numerous methods are known for preparing microparticles of
any particular size range. In the various delivery vehicles of the
present invention, the microparticle sizes may range from about 0.2
.mu.m up to about 100 .mu.m. Synthetic methods for gel
microparticles, or for microparticles from molten materials are
known, and include polymerization in emulsion, in sprayed drops,
and in separated phases. For solid materials or preformed gels,
known methods include wet or dry milling or grinding,
pulverization, size separation by air jet, sieve, and the like.
[0086] Microparticles can be fabricated from different polymers
using a variety of different methods known to those skilled in the
art. Exemplary methods include those set forth below detailing the
preparation of polylactic acid and other microparticles. Polylactic
acid microparticles are preferably fabricated using one of three
methods: solvent evaporation, as described by Mathiowitz, et al.
(1990) J. Scanning Microscopy 4:329; Beck, et al. (1979) Fertil.
Steril. 31: 545; and Benita, et al. (1984) J. Pharm. Sci. 73: 1721;
hot-melt microencapsulation, as described by Mathiowitz, et al.,
Reactive Polymers 6: 275 (1987); and spray drying. Exemplary
methods for preparing microencapsulated bioactive materials are set
forth below.
[0087] In the solvent evaporation method, the microcapsule polymer
is dissolved in a volatile organic solvent, such as methylene
chloride. The green tea polyphenol (either soluble or dispersed as
fine particles) is added to the solution, and the mixture is
suspended in an aqueous solution that contains a surface active
agent such as poly(vinyl alcohol). The resulting emulsion is
stirred until most of the organic solvent has evaporated, leaving
solid microparticles. The solution is loaded with the green tea
polyphenol and suspended in vigorously stirred distilled water
containing poly(vinyl alcohol) (Sigma). After a period of stirring,
the organic solvent evaporates from the polymer, and the resulting
microparticles are washed with water and dried overnight in a
lyophilizer. Microparticles with different sizes (1-1000 .mu.m) and
morphologies can be obtained by this method. This method is useful
for relatively stable polymers like polyesters and polystyrene.
Labile polymers such as polyanhydrides, may degrade during the
fabrication process due to the presence of water. For these
polymers, the following two methods, which are performed in
completely anhydrous organic solvents, are preferably used.
[0088] In the hot melt encapsulation method, the polymer is first
melted and then mixed with the solid particles of biologically
active material that have preferably been sieved to less than 50
microns. The mixture is suspended in a non-miscible solvent (like
silicon oil) and, with continuous stirring, heated to about 5.
degree. C. above the melting point of the polymer. Once the
emulsion is stabilized, it is cooled until the polymer particles
solidify. The resulting microparticles are washed by decantation
with a solvent such as petroleum ether to give a free-flowing
powder. Microparticles with sizes ranging from about 1 to about
1000 microns are obtained with this method. The external surfaces
of capsules prepared with this technique are usually smooth and
dense. This procedure is preferably used to prepare microparticles
made of polyesters and polyanhydrides.
[0089] The solvent removal technique is preferred for
polyanhydrides. In this method, the green tea polyphenol is
dispersed or dissolved in a solution of the selected polymer in a
volatile organic solvent like methylene chloride. This mixture is
suspended by stirring in an organic oil (such as silicon oil) to
form an emulsion. Unlike solvent evaporation, this method can be
used to make microparticles from polymers with high melting points
and different molecular weights. Microparticles that range from
about 1 to about 300 .mu.m can be obtained by this procedure. The
external morphology of spheres produced with this technique is
highly dependent on the type of polymer spray drying, the polymer
is dissolved in methylene chloride. A known amount of the green tea
polyphenol is suspended or co-dissolved in the polymer solution.
The solution or the dispersion is then spray-dried. Microparticles
ranging between about 1 to about 10 .mu.m are obtained with a
morphology which depends on the type of polymer used.
[0090] In one embodiment, the green tea polyphenol is encapsulated
in microcapsules that comprise a sodium alginate envelope.
Microparticles made of gel-type polymers, such as alginate, are
produced through traditional ionic gelation techniques. The
polymers are first dissolved in an aqueous solution, mixed with
barium sulfate or some bioactive agent, and then extruded through a
microdroplet forming device, which in some instances employs a flow
of nitrogen gas to break off the droplet. A slowly stirred
(approximately 100-170 RPM) ionic hardening bath is positioned
below the extruding device to catch the forming microdroplets. The
microparticles are left to incubate in the bath for about twenty to
thirty minutes in order to allow sufficient time for gelation to
occur. Microparticle size is controlled by using various size
extruders or varying either the nitrogen gas or polymer solution
flow rates.
[0091] Liposomes can aid in the delivery of the green tea
polyphenol to a particular tissue and also can increase the
half-life of green tea polyphenol. Liposomes are commercially
available from a variety of suppliers. Alternatively, liposomes can
be prepared according to methods known to those skilled in the art,
for example, as described in Eppstein et al., U.S. Pat. No.
4,522,811. In general, liposomes are formed from standard
vesicle-forming lipids, which generally include neutral or
negatively charged phospholipids and a sterol, such as cholesterol.
The selection of lipids is generally guided by consideration of
factors such as the desired liposome size and half-life of the
liposomes in the blood stream. A variety of methods are known for
preparing liposomes, for example as described in Szoka et al., Ann.
Rev. Biophys. Bioeng. 9: 467 (1980); and U.S. Pat. Nos. 4,235,871,
4,501,728, 4,837,028, and 5,019,369. In one embodiment the
liposomes encapsulating the green tea polyphenol comprise a ligand
molecule that can target the liposome to a particular cell or
tissue at or near the site of psoriasis. Ligands which bind to
receptors prevalent in eplithelial tissue, such as monoclonal
antibodies that bind to epithelial tissue,
[0092] In one embodiment, the liposomes encapsulating the green tea
polyphenols of the present disclosure are modified so as to avoid
clearance by the mononuclear macrophage and reticuloendothelial
systems, for example by having opsonization-inhibition moieties
bound to the surface of the structure. In one embodiment, a
liposome can comprise both opsonization-inhibition moieties and a
ligand. Opsonization-inhibiting moieties for use in preparing the
liposomes in one embodiment are large hydrophilic polymers that are
bound to the liposome membrane. As used herein, an opsonization
inhibiting moiety is "bound" to a liposome membrane when it is
chemically or physically attached to the membrane, e.g., by the
intercalation of a lipid-soluble anchor into the membrane itself,
or by binding directly to active groups of membrane lipids. These
opsonization-inhibiting hydrophilic polymers form a protective
surface layer which significantly decreases the uptake of the
liposomes by the macrophage-monocyte system ("MMS") and
reticuloendothelial system ("RES"); e.g., as described in U.S. Pat.
No. 4,920,016. Liposomes modified with opsonization-inhibition
moieties thus remain in the circulation much longer than unmodified
liposomes. For this reason, such liposomes are sometimes called
"stealth" liposomes. Stealth liposomes are known to accumulate in
tissues fed by porous or "leaky" microvasculature. In addition, the
reduced uptake by the RES lowers the toxicity of stealth liposomes
by preventing significant accumulation in the liver and spleen.
[0093] Opsonization inhibiting moieties suitable for modifying
liposomes are preferably water-soluble polymers with a molecular
weight from about 500 to about 40,000 daltons, and more preferably
from about 2,000 to about 20,000 daltons. Such polymers include
polyethylene glycol (PEG) or polypropylene glycol (PPG)
derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate;
synthetic polymers such as polyacrylamide or poly N-vinyl
pyrrolidone; linear, branched, or dendrimeric polyamidoamines;
polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and
polyxylitol to which carboxylic or amino groups are chemically
linked, as well as gangliosides, such as ganglioside GMI.
Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives
thereof; are also suitable. In addition, the opsonization
inhibiting polymer can be a block copolymer of PEG and either a
polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine,
or polynucleotide. The opsonization inhibiting polymers can also be
natural polysaccharides containing amino acids or carboxylic acids,
e.g., galacturonic acid, glucuronic acid, mannuronic acid,
hyaluronic acid, pectic acid, neuraminic acid, alginic acid,
carrageenan; laminated polysaccharides or oligosaccharides (linear
or branched); or carboxylated polysaccharides or oligosaccharides,
e.g., reacted with derivatives of carbonic acids with resultant
linking of carboxylic groups. Preferably, the
opsonization-inhibiting moiety is a PEG, PPG, or derivatives
thereof. Liposomes modified with PEG or PEG-derivatives are
sometimes called "PEGylated liposomes," The opsonization inhibiting
moiety can be bound to the liposome membrane by any one of numerous
well-known techniques. For example, an N-hydroxysuccinimide ester
of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble
anchor, and then bound to a membrane. Similarly, a dextran polymer
can be derivatized with a stearylamine lipid-soluble anchor via
reductive amination using Na(CN)BH3 and a solvent mixture such as
tetrahydrofuran and water in a 30:12 ratio at 60.degree. C.
[0094] The disclosed microparticles and liposomes and methods of
preparing microparticles and liposomes are offered by way of
example and are not intended to define the scope of microparticles
or liposomes of use in the present disclosure. It will be apparent
to those of skill in the art that an array of microparticles or
liposomes, fabricated by different methods, are of use in the
present invention.
Materials and Methods
[0095] Chemicals and antibodies: EGCG was purchased from
Sigma-Aldrich (St. Louis, Mo.). Anti-human CENP-C (H-300) Rabbit
polyclonal antibody, Anti-human 52 KD Ro/SSA (D-12) mouse
monoclonal antibody, anti-human PARP (F-2) mouse monoclonal
antibody, anti-human p38, pp 38, pJNK and pERK antibodies, and
anti-human actin (1-19) goat polyclonal antibody were purchased
from Santa Cruz Biotechnology, Santa Cruz, Calif. The anti-human
coilin mouse monoclonal antibody was obtained from By Transduction
Laboratories, San Jose, Calif. The anti-human Golgin-67 rabbit
polyclonal antibody was a kind gift from Dr. Don Fujita, University
of Calgary, Canada. The anti-human La/SSB mouse monoclonal antibody
was purchased from Immunovision Diagnostics Inc., Springdale, Ark.
The mouse Anti-Nuclear Antibodies (ANA) ELISA Kit was purchased
from Alpha Diagnostic International, Inc. San Antonio, Tex.
Specific inhibitors for p38 (SB 203580), JNK (Sp600125) and ERK (PD
98059) were supplied by EMD Bioscience, Inc., San Diego, Calif. The
70% GTPs are provided by Zhejinag Cereals, Oils & Foodstuffs
Imp/Exp Co., Ltd, China, which contain 40% EGCG, 13% ECG, 7.3% EGC,
3.2% EC, and 2.7% Caffeine. Animal treatment. The NOD mice were
purchased from Jackson Laboratory. The NOD/LtJ strain develops
spontaneous autoimmune symptoms that resemble human SS (Cha, S. et
al. (2002) Crit. Rev Oral Biol Med. 13:4-16). The NOD mouse is an
important model system that has provided clues to the cellular
mechanisms involved in SS. Almost 50% of published animal studies
for SS used this model during the past two years. This mouse strain
develops a lymphocytic infiltration of exocrine tissues at 10-12
weeks of age, particularly in females, and was originally used as a
model for type I diabetes. NOD-scid congenic mice (that lack
functional lymphocytes) do not develop sialadenitis (or diabetes).
However, they do show dysfunction in expression of biochemical
markers of salivary gland differentiation such as amylase and
parotid secretory protein (PSP). These data are consistent with a
model of SS in which there is an initial phase during which
dysregulation of glandular homeostasis triggers the disease,
followed by an immune cell-mediated phase that leads to a loss of
secretory function. All animal protocols in this study were
approved by the Institutional Animal Care and Use Committee.
Animals (two groups, 15 NOD mice/group) were allowed ad libitum
access to either water or 0.2% GTPs starting at the 9.sup.th week
of age. After the onset of autoimmune disease (determined as
diabetes, detected by Glucotest strips), each animal was allowed to
progress with the disease for three weeks, and then euthanized (2
water-fed control mice died during this 3-week period). Blood was
collected from each animal in the above described treatment groups
by cardiac puncture immediately after euthanasia, and serum for
ELISA assays was prepared by centrifugation of blood samples at
3000 rpm. The submandibular glands were dissected free of the
sublingual gland and attached tissues for pathological
analyses.
[0096] Determination of serum total autoantibodies: Samples from
the two groups were examined by ELISA assays for anti-SS-associated
autoantibodies using the Mouse Anti-Nuclear Antibodies (ANA) ELISA
Kit (Cat #5200, Alpha Diagnostic International, Inc. San Antonio,
Tex.) according to the manufacturer's instructions. This kit
detects total ANA against ds-DNA, ss-DNA, histones,
ribonucleoproteins (RNPs), SS-A, SS-B, SM antigens, Jo-1, and
Scl-70. Samples were analyzed with blanks, positive and negative
controls in 96-well plates by ELISA reaction, photo-detection using
a VERSAmax Microplate Reader at 450 nm, and statistical analysis
using two-tailed student t-test.
[0097] Immunohistochemistry: The submandibular glands from NOD mice
were fixed in 10% neutral-buffered formalin, paraffin embedded,
sectioned at 5 [mu]m, and stained with H&E by routine methods
previously described (Hsu et al, 2005).
[0098] Pathology scoring of lymphocyte infiltration in the
submandibular glands of NOD mice, We adopted the cumulative focus
score (cFS) criteria recently published by Morbini et al. (101) for
the assessment of salivary gland inflammatory infiltrates as a
component of the diagnosis of Sjogren's syndrome (SS). Briefly,
these criteria modify the American-European Consensus Group (Vitali
et al, 2002) focus score (FS) criterion as a component of the
diagnosis of SS by adopting a multilevel sectioning and evaluation
of salivary gland tissues of suspected SS patients. This multilevel
sampling improved the diagnostic accuracy of biopsies with a
baseline FS between 1 and 2, which represents the critical cutoff
in SS histopathological evaluation (Morbini et al, 2005). The cFS
method assesses the number of chronic (lymphocytic) inflammatory
cell infiltrates of at least 50 in a 10-HP (equivalent to 4
mm.sup.2) light microscopy field repeated for a minimum of three
different tissue section levels. The arithmetic average FS from all
examined levels from a particular gland represents the cFS average
for that gland. To examine differences in the size of foci above 50
cells, a quantitative analysis was performed using the BIOQUANT
NOVA PRIME 6.75 software. One H&E-stained submandibular
salivary gland section was selected at random for each of the
animals in the two groups and images of areas containing foci were
loaded into the software. The areas of lymphocyte infiltration foci
were captured individually and measured quantitatively by the soft
ware as relative density units.
[0099] Cell lines: NHEK were purchased from Cambrex (East
Ruthtrford, N.J.) and sub-cultured in KGM-2 provided by the
manufacturer. Subculture of the NHEK was performed by detaching the
cells in 0.25% trypsin, and transferring into new tissue culture
flasks. The immortalized human salivary gland acinar cells NS-SV-AC
have been previously described (Azuma M. et al. (1993) Lab Invest.
69(1):24-42). These cells were selected following transfection of
origin-defective SV40 mutant DNA and maintained in KOM-2 medium,
they were kindly provided by Dr. Masayuki Azuma (Tokushima
University School of Dentistry, Tokushima, Japan). Subculture of
the cells was performed by detaching the cells in 2.5% trypsin, and
transferring into new tissue culture flasks.
[0100] Cell treatment: ECCG was dissolved in cell culture media as
a 50 mM stock immediately before use. Exponentially growing
NS-SV-AC cells were incubated with 100 .mu.M ECCG for various time
periods in the growth media described above, and then were either
extracted for total RNA or cell lysates prior to RT-PCR or Western
blots.
Affymetrix Gene Array Analyses
[0101] The gene array experiment was performed at the Core Facility
of Genomics of the Medical College of Georgia, according to the
instructions of the manufacturer (Affymetrix, Santa Clara, Calif.).
Briefly, NHEK and OSC2 cells treated with 100 .mu.M EGCG for
different time period were extracted for total RNA, the RNA was
processed for probes and hybridized with a cDNA microarray
(HuG133A, Affymetrix) which represents 22,283 human series. DNA
chips were read with a Hewlett-Packard GeneArray Scanner at a
resolution of 3 mm and were analyzed with the GeneSpring software
(Silicon Genetics, Redwood City, Calif.). Details of this analysis
will be published elsewhere (Hsu et al., manuscript in
preparation).
Total RNA Extraction and Semi-Quantitative Reverse-Transcription
PCR (KT-PCR)
[0102] Total RNA was extracted using an RNeasy total RNA isolation
system (QIAGEN, Valentia, Calif.) RT reactions and PCR reactions
were performed according to the manufacturer's protocol (Super
Array Bioscience Corp). For amplification, the following pair of
primers was used,
TABLE-US-00001 GAPDH: Sense, 5'-TCCCATCACCATCTTCCA-3', (SEQ ID NO.
1) Antisense, 5'-CATCACGCCACACGAGTTTCC-3', (SEQ ID NO: 2) SS-A/Ro
(469 bp): sense 5'-GAACTGCTGCAGGAGGTGATAA-3', (SEQ ID NO: 3)
Antisense 5'-GGCACATTCAGAGAAGGAGT-3', (SEQ ID NO: 4) SS-B/La (95
bp): sense 5'-CCAAAATCTGTCATCAAATTGAGTATT-3', (SEQ ID NO: 5)
Antisense 5'-CCAGCCTTCATCCAGTTTTATCT-3', (SEQ ID NO: 6)
[0103] Amplification was started by heating for 1.5 min at
94.degree. C., followed by 30 cycles for 52 kD SSA/Ro. 25 cycles
for GAPDH and 25 cycles for SSB/La, each cycle consisting of 15
second at 94.degree. C., 30 seconds at 57.3.degree. C., and 1 min
at 72.degree. C. A final extension was performed at 72.degree. C.
for 5 min prior to gel analysis.
[0104] Western blot. Cells were washed in ice-cold PBS and lysed
for 20 min in RIPA buffer containing 1% (v/v) Nonidet P-40, 0.5%
(w/v) sodium deoxycholate, 0.1% (w/v) SDS, 10 .mu.g/ml leupeptin, 3
.mu.g/ml aprotinin and 100 mM phenylmethylsulfonyl fluoride (PMSF).
Samples of lysates containing the same amount of protein were
loaded in each lane (we used 5-.mu.g, depending on the antibody
used) and electrophoretically separated on a 12% SDS polyacrylamide
gel. Following electrophoresis, proteins were transferred to a PVDF
membrane (Immobilon.TM.-P, Millipore Corporation, Bedford, Mass.).
The membrane was blocked for 1 h with 5% (w/v) non-fat dry milk
powder in PBST (0.1% Tween-20 in PBS) and then incubated for 1 h
with primary antibody diluted in PBST/milk (Antibodies and
dilutions: rabbit polyclonal JNK1/2, 1:000, mouse monoclonal
pJNK1/2, 1:1000, rabbit polyclonal ERK, 1:2000, rabbit polyclonal
p38, 1:1000, rabbit polyclonal pp 38, 1:1000 and goat polyclonal
actin, 1:2000). The membrane was washed three times with PBST and
incubated with peroxidase-conjugated, affinity-purified anti-rabbit
IgG (Santa Cruz Biotechnology, Inc.) for 1 h. Following extensive
washing, the reaction was developed by enhanced chemiluminescent
staining using ECL Western blotting detection reagents (Amersham
Pharmacia Biotech Inc., Piscataway, N.J.).
[0105] TNF-.alpha. cyotoxicity: NS-SV-AC cells
(0.5.times.10<4> cells/well) were seeded in a 96-well
microplate and either treated for 24 hr with EGCC as described
above (or not treated in the control), in the presence of 100 ng/ml
TNF.alpha. and 10 .mu.g/ml cyclohexamide. After the treatments,
viability was determined by the MTT assay. The cells in each well
were washed with 200 .mu.l of phosphate-buffered saline (PBS) and
incubated with 100 .mu.l of 2% (w/v) MTT in a solution of 0.05 M
Tris-HCl (pH 7.6), 0.5 mM MgCl.sub.2, 2.5 mM CoCl.sub.2 and 0.25 M
disodium succinate at 37.degree. C. for 30 min. Cells were fixed by
the addition of 100 .mu.l of 4% (v/v) formalin in 0.2 M Tris-HCl
(pH 7.6), and after a 5 min incubation at room temperature liquid
was removed and the wells were allowed to dry. Each well was rinsed
with 200 .mu.l water and cells were solubilized by the addition of
100 .mu.l of 6.35% (v/v) 0.1 N NaOH in DMSO. The colored formazan
product was measured by a Thermo MAX micro plate reader (Molecular
Devices Corp. Sunnyvale, Calif.) at a wavelength of 562 nm.
EXAMPLES
Example 1
Affymetrix Gene Array Analyses
[0106] Gene assays provide a valuable tool for studying the broad
response of cells to agents, and we have used this approach to
compare the differential effects of GTPP on gene expression in NHEK
and human oral squamous carcinoma cells (Hsu, et al, manuscript in
preparation). The results demonstrated that approximately 2100
genes were either up- or down-regulated by at least 2-fold in
response to 100 uM EGCG in both cell types during the 24 hrs
post-treatment. Examination of the relative NHEK mRNA levels of
genes represented on the chip encoding autoantigens previously
identified in SLE (Sherer et al., 2004) revealed a 2-fold or
greater change in expression of a number of genes during the 24 hr
post-treatment time course (Table 1). See also Hsu, S. et al.
(2005) JPET 315:805-811, which is incorporated by reference in its
entirety.
[0107] Several patterns of change were observed. For example, the
nuclear autoantigen genes (RNA polymerase I and SS-B/La), a
cytoskeletal autoantigen gene (alpha-fodrin) and a Golgi
autoantigen gene (golgin-67) showed a general pattern of rapid
(0.5-2 hr initiation) and persistent 2-fold or greater decrease in
mRNA levels during the 24 hr period. Other genes showed an initial
decrease during the first 2 hrs (e.g., centromere protein Cl,
coilin), with levels returning to near normal by 24 hrs. Other
autoantigen genes (e.g., nuclear antigen SPIOO and scleroderma
autoantigen 1) showed a rapid initial increase (2-4-fold) followed
by a 2-fold or greater decrease. Other genes (e.g., one of the the
arrayed Ku autoantigen genes) showed a transient initial increase,
with levels returning to near normal. In contrast, other
antoantigen genes did not show a significant change during the time
course of the experiment. For example, 60 kDa SS-A/Ro did not
decline significantly (52 kDa SS-A/Ro cDNA was not included in the
gene array).
TABLE-US-00002 TABLE 2 Summary of gene array analysis of
autoantigen gene expression. Analysis of the gene array dataset for
EGCG-treated normal human epidermal keratinocytes (NHEK) cells
demonstrates a general pattern of reduction in autoantigen gene
expression. Time 0, Time 0.5, Time 02, Time 06, Times 24,
Affymetrix Cell Type Cell Type Cell Type Cell Type Cell Type cDNA
Array NHEK NHEK NHEK NHEK NHEK Genes coding Symbols normalized
normalized normalized normalized normalized for autoantigens
20473_at 1 1.27 0.46 0.87 0.94 centromere protein C1 203653_s_at 1
0.49 0.54 0.60 0.79 coilin 203654_s_at 1 1.11 0.78 0.62 0.79 coilin
208797_s_at 1 1.26 0.71 0.92 0.29 golgin-67 208798_x_at 1 0.81 0.46
0.32 0.31 golgin-67 210425_x_at 1 0.50 0.47 0.19 0.31 golgin-67
213650_at 1 0.24 0.61 0.69 0.72 golgin-67 200792_at 1 1.15 1.19
1.13 0.79 thyroid autoantigen 70 kDa (Ku antigen) 208642_s_at 1
1.29 1.14 0.96 0.73 Ku autoantigen, 80 kDa 208643_s_at 1 2.34 1.40
0.98 0.77 Ku autoantigen, 80 kDa 202692_s-at 1 0.50 0.48 0.50 0.55
RNA polymerase 1 208611_s_at 1 0.41 0.75 0.56 0.42 fodrin-.alpha.
212071_s_at 1 0.91 0.92 0.95 0.72 spectrin-.beta. non-
erythrocyctic 1 215235_s_at 1 0.49 0.97 0.75 0.71 fodrin-.alpha.
212852_s_at 1 0.60 0.98 0.85 0.94 60 kDa, SS-A/Ro 210438_x_at 1
0.71 0.92 0.69 0.88 60 kDa, SS-A/Ro 201139_s_at 1 0.71 0.74 0.43
0.26 SS-B/La 201138_s_at 1 0.84 0.88 0.57 0.27 SS-B/La 202863_at 1
1.72 1.04 0.59 0.36 Nuclear antigen SP100 202864_s_at 1 3.12 1.21
0.64 0.08 Nuclear antigen SP100 213226_at 1 3.99 1.09 0.43 0.50
Nuclear antigen SP10075 kDa 213226_at 1 3.99 1.09 0.43 0.50
scleroderma autoantigen 1, 75 kDa
[0108] An additional mechanism by which EGCG modulation of gene
expression could afford protection in autoimmune disorders might be
by reduction in the expression of proinflammatory signaling
molecules. Data show that there is a broad reduction in expression
of pro-inflammatory genes. Table 3 shows the results for a number
of improtant inflammation-related genes whose expression in NHEK is
modulated by EGCG (the entire group is too large to display). These
data show that EGCG can suppress the expression of many
inflammatory factors. Importantly, the MAPK family member p38 is
induced.
TABLE-US-00003 TABLE 3 Selected inflammatory gene expression
detected from NHEK treated with 100 .mu.M EGCG for indicated hours.
Except p38 and IL8, all other genes were inhibited by EGCG. Numbers
represent fold up/down at the time points indicated. Time 0.5, Time
02, Time 06, Times 24, Time 0, Cell Cell Type Cell Type Cell Type
Cell Type Affymetrix Common Type NHEK NHEK NHEK NHEK NHEK symbols
Names normalized normalized normalized normalized normalized
205067_at IL1B 1 0.29 0.58 0.49 0.26 39402_at IL1B 1 0.29 0.48 0.40
0.27 210118_s_at IL1A 1 0.66 0.42 0.54 0.27 205290_s_at BMP2 1 0.32
0.96 0.72 0.44 206295_at IL18 1 0.33 0.65 0.70 0.65 206172_at
IL13RA2 1 0.33 0.74 0.81 0.55 21100_s_at IL6ST 1 0.34 0.28 0.99
0.61 209575_at IL10RB 1 0.43 0.63 0.76 0.38 205945_at IL6R 1 0.48
0.69 0.31 0.69 221085_at TNFSF15 1 0.52 1.56 0.67 0.63 202727_s_at
IFNGR1 1 0.79 0.91 0.45 0.92 202859_x_at IL8 1 6.02 9.55 9.99 9.93
21156_x_at p38 1 2.32 1.46 1.62 2.51 202530_at p38 1 2.51 1.09 0.63
1.17
Example 2
Semi-Quantitative RT-PCR
[0109] The preliminary array analysis described above provided data
suggesting that GTPP could alter the expression of some genes
encoding autoantigens. To further test this possibility, three cell
types, NHEK, NS-SV-AC (an SV40 immortalized cell line derived from
human submandibular acinar cells) and OC2 cells (derived from an
oral squamous cell carcinoma) were treated with 100 uM EGCG for
different times. A semi-quantitative estimate of SS-A/Ro and
SS-B/La mRNA levels was then obtained by RT-PCR during the
exponential period of amplification. GAPDH was used as a
housekeeping gene control. SS-B/La and ES-A/Ro 52 were selected
because elevated mRNA levels (2-3 fold) of SS-A/RO 52 and SS-B/La
are found in salivary tissues of SS patients (Bolstad A. I., et al.
(2003) Arthritis Rheum 48:174-185), and autoantibodies against
SS-A/Ro end SS-B/La are found in nearly all (about 95% and 87%,
respectively) primary SS patients (Hahn: 1998). Consistent with the
gene array data, SS-R/La message decreased progressively and
substantially in both NHEK and NS-SV-AC cells (FIG. 1). The levels
of SS-A/Ro (52 kd) mRNA also showed a reduction during treatment,
although the effect was less pronounced than that seen for SS-B/La
and was not prominent until 24 hrs. In OSC2 cells the reduction in
SS-B/La mRNA was less pronounced and did not occur until 24 hrs of
exposure, while SS-A/Ro mRNA showed little change. GAPDII also
showed no marked decrease in these cells in response to EGCG,
indicating the reduction in mRNA levels seen for SS-B/La and
SS-A/Ro was not due to a generalized effect on the cells.
Example 3
Protein Levels of Autoantigens
[0110] To extend the mRNA data, the protein levels of 6 different
autoantigens in NHEK and NS-SV-AC cells were determined by Western
analysis following two treatments with 100 uM EGCSG at 24 and 48
hrs. As shown in FIG. 2, coilin and PARP protein levels were
significantly reduced in both cell lines by 24 hrs, and were barely
detectable after 48 hrs. CENP-C showed a similar trend, although
the reduction was less pronounced. Neither SS-A/Ro 52 nor SS-B/La
were significantly reduced in either cell line by 24 hrs, but were
considerably reduced by 48 hrs. Golgin-67 was reduced in NS-SV-AC
cells by 24 hrs, and barely detectable at 48 hrs. Golgin-67 was
also reduced in NHEK cells, although the reduction was not as
marked and was only observed at 48 hrs. Actin protein levels were
unchanged by EGCG during the 48 hr treatment period.
Example 4
Serum Total Autoantibody ELISA
[0111] NOD mice were fed either water or water containing 0.2% GTPs
for 3 weeks. Serum of 27 animals was analyzed: 15 from the
GTP-water and 12 from the water-only group (two animals died, one
animal was not able to collect serum in the latter group). There
was a significant difference between the total serum antibody
levels from the GTP-water group and water-only group. On average,
the total ANA (against ds-DNA, ss-DNA, histones, ribonucleoproteins
[RNPs], SS-A, SS-B, SM antigens, Jo-1, and Scl-70) in the GTP-water
animal were approximately 20% lower than that of the water-only
animals (FIG. 3). This result indicates that oral administration of
GTPs significantly reduced the serum autoantibody levels
(two-tailed student t-test, p=0.036, n=27).
Example 5
Analysis of Lymphocyte infiltration
[0112] The submandibular glands of each NOD animal were collected
and the standardized scores for the inflammatory cell infiltrates
were determined blindly, as described in the methods. FIG. 4 shows
representative submandibular glands from a water-fed control (A)
and a GTP/water-fed NOD mouse (3). Pathological focal scoring,
using the cumulative focus score (cFS) criteria for SS diagnosis,
demonstrated no significant difference in focal scores (i.e. the
number of focal inflammatory cell aggregates containing 50 or more
lymphocytes in each 4 mm.sup.2 area) between the GTP-treated and
untreated (water) controls. The average focal score was
2.125.+-.1.13 for GTP-fed mice and 2.125.+-.0.64 for control mice.
However, inspection of the foci suggested potential differences in
the focal areas between the groups, equivalent to differences in
the total number of inflammatory cells/focus. For example, both
animals shown in FIG. 4 received a focal score of 3, but the foci
in the GTP-treated animal appear smaller (although for any one
focal group this might just reflect an off-center cut through its
volume). Quantitative analysis of the areas of lymphocyte
infiltration foci in H&E-stained submandibular gland sections
(FIG. 5, n=27 animals) demonstrated a statistically significant
difference (p=0.006, two-tailed t-test, n=83 foci/group) between
the groups in the number of inflammatory cells/infiltrate, with
fewer cells in the salivary glands of GTPs/water-fed animals.
Example 6
Human Salivary Gland Acinar Cells are Protected from
TNF-.alpha.-Induced Cytotoxicity by EGCG
[0113] TNF-.alpha., which is produced by inflammatory cells, is
known to induce cytotoxicity in many cell types, and can be
down-regulated by EGCG (Suganuma et al, 2000, Fujiki et al, 2000,
Fujiki et al, 2003). Therefore, one mechanism by which EGCG could
ameliorate the effects of SS could be attenuation of
TNF-.alpha.-cytotoxicity. We examined the effects of EGCG on
TNF-.alpha.-induced cytotoxicity of the human salivary gland acinar
cell line NS-SV-AC using the MTT assay. Results from these
experiments are summarized in FIG. 6. EGCG in a dose-dependent
manner provided significant protection of NS-SV-AC cells (up to
50%) from TNF.alpha.-induced cytotoxicity (two-tailed t-test,
p<0.05).
Example 7
p38 MAPK Inhibitor SB203580 and MEK Inhibitor PD98059 Abolished the
Protective Effect of EGCG
[0114] To examine the role of activation of the p38 signaling
pathway in reducing TNF-.alpha.-induced cytotoxicity, cells were
exposed to TNF-[alpha] and EGCG in the presence of either the p38
MAPK inhibitor SB203580 or the MEK inhibitor PD98059. The results
of MTT assays indicated that both inhibitors significantly reduced
the protective effect of EGCG against TNF-.alpha.-induced loss of
cell viability (FIG. 7).
Example 8
EGCG Specifically Activates the Phosphorylation of P38 in NS-SV-AC
Cells
[0115] p38 activation is one mechanism by which GTPs attenuates
mechanisms of SS pathogenesis. As shown in FIG. 8, EGCG induced a
rapid and sustained phosphorylation of p38 in NS-SV-AC cells while,
only a slight increase in pERK was detected, and phosphorylation of
JNK was not altered. Therefore, of these three signaling pathways,
EGCG activates primarily p38 in NS-SV-AC cells.
[0116] In the NOD mouse model of SS, oral administration of GTPs
lowered the total serum autoantibody level and reduced the
magnitude of salivary lymphocyte infiltration. In vitro, EGCC
protected acinar-derived cells from TNF-.alpha.-induced
cytotoxicity. These protective effects of GTPs were associated with
the p38 MAPK-signaling pathway.
Example 9
Pathology Scoring of Lymphocyte Infiltration in the Submandibular
Glands of Early GTP-Treated MRL and NOD Mice
[0117] FIG. 9 shows representative sections of H&E
submandibular glands from each experimental group (disease free,
diseased animals treated with GTP, and diseased untreated animals)
in both strains, together with the corresponding inflammatory
infiltrate score. As shown in FIG. 9, glands from disease-free
animals showed cFS of 0; disease plus GTPs a cFS of 1 and 2; and
disease a cFS of 2.5 and 3, for NOD and MRL, respectively. These
data show a marked in vivo effect of GTPs on the progression and
severity of SS-like pathology. Both MRL and NOD mice showed a
reduction in focal score, suggesting GTPs effects are not strain
specific.
TABLE-US-00004 TABLE 1 cDNA microarray determination of autoantigen
expression in NHEK treated with 100 .mu.M EGCG for the indicated
hours. Numbers represent fold up/down at the time points normalized
to time zero levels. 0 0.6 2 6 24 Autoantigen h Descriptions
Inhibited > 2-fold AKAP12 1 0.808 0.891 0.4 0.188 A kinase
IPREA1 anchor protein igmarion 12 .alpha.-NAC 1 0.082 0.521 0.0143
0.189 Homo sapiens .alpha.-NAC gene for nascent polypeptide-
associated complex component CENPB 1 0.803 0.773 0.4 8 0.721
Centomero protein B CENPO3 1 1.260 0.457 0.880 0.021 Centomero
protein C1 FL131657 1 0.332 0.003 0.435 0.009 Hypothetical protein
FL131657 FLNR 1 0.78 0.86 0.875 0.2 Filamin B, y (actin-binding
protein 78) FLN 1 1.279 1.168 0.751 0.249 Filamin B, B
(actin-binding protein 378) GOLGA1 1 0.289 1.122 0.08 0.9 Golgi
autoantigen, golgin subfamily a, 1 GOLGA2 1 0.822 0.012 0.709 0.219
Golgi autoantigen, golgin subfamily a, 2 GOLGA3 1 0.833 0.68 0.617
0.419 Golgi autoantigen, golgin subfamily a, 3 GOLGA4 1 0.822 0.748
0.005 0.031 Golgi autoantigen, golgin subfamily a, 4 HSAC01 1 0.158
0.7 0. 2 0.562 Nucleolar cystoine-rich protein HUMAUNTIC 1 0.618
0.779 0.114 0.517 Nucleolar GTPase LMO4 1 0.72 0.807 0.706 1.011
LIM damelts only 4 POLB2A 1 0.12 1.29 1.90 1.02 Polysperace (RNA II
(DNA-directed) polypeptide A, 220 kDa POLB2A 1 1.09 0.45 0.41 1.17
Polysperace (RNA II (DNA-directed) polypeptide A, 220 kDa POLB2D 1
1.14 0. 1.01 0.29 Polysperace (RNA II (DNA-directed) polypeptide D
POLB2E 1 0.45 0. 0.37 0.39 PolypeptideE, 25 kDa POLB30 1 0.20 0.78
0.43 0.49 Polysperace (RNA III (DNA-directed) (32 kDa) POLB20 1
1.26 1.00 1.05 0.47 Polysperace (RNA III (DNA-directed) (32 kDa)
SNRPA 1 1. 0.89 1.05 0.33 Small nuclear ribonucleoprotein
polypeptide A SP100 1 1. 1.087 0.59 0.956 Nuclear antigen Sp100
SPTAN1 1 0. 0.75 0.53 0.42 Spectrin, .alpha., 1 ( ) SPTAN1 1 0.
0.07 0.75 0.71 Spectrin, .alpha., 1 ( ) SNRPB 1 0.56 0.08 0.78 0.59
Small nuclear ribonucleoprotein polypeptide B and B1 SNRPB 1 0.02
0.70 0.67 0.42 Small nuclear ribonucleoprotein polypeptide B and B1
SNRPB2 1 0.49 0.07 0.50 0.82 Small nuclear ribonucleoprotein
polypeptide B* SSA2 1 1.009 0.035 0.739 1.17 1F1 NIH_M3O_03 77, H.
sapiens cDNA clone IMAGE: 4071854 5', mRNA sequence SS 1 0.833
0.879 0.57 0.273 antigen B SS 1 0.711 0.729 0.427 0.257 antigen B
STRN3 1 0.4 5 0.029 0.823 0.7 9 , binding protein 9 TOP1 1 0.88
1.05 0.51 0.12 Topoiso (DNA) I TOP1 1 0.63 0.02 0.03 0.12 Topoiso
(DNA) I URTF 1 0.493 0.484 0.301 0.848 Upstrenin binding
transcription factor, RNA poly I Induced then inhibited > 2-fold
PMSOLL 75 kDa 1 8.993 1.09 0.481 0.428 Poly autoantigen 1 PSME 1
2.551 1.151 1.128 0.421 subunit SNRPD1 1 2.098 1.255 0.058 0.405
Small nuclear ribonucleoprotein D1 polypeptide, 18 kDa SP100 1
8.128 1.305 0.093 0.070 Nuclear antigen Sp100 Induced then declined
to control levels ADPRT 1 3.547 1.201 0.992 0.85
ADP-ribosyltransferase [NAD.sup.+, poly polymerase] CENPA 1 7.077
1.095 0.672 0.073 Centromere protein A, 17 kDa MCCSSO 1 1.056 1.101
1.618 0.81 Hypothetical protein NOC5560 MCCSSO 1 7.07 0.332 2.805
0.002 Hypothetical protein NOC5560 SP110 1 3.735 1.702 1.205 0.975
SP110 nuclear body protein SP110 1 4.178 1.804 0.007 0.054 SP110
nuclear body protein SNRPT0 1 3.59 1.01 2.28 0.51 Small nuclear
ribonucleoprotein 70-kDa polypeptide (EN antigen) XRCC5 1 2.328
1.403 0.577 0.771 X-ray repair complementing deflective repair in
Chinase beveator cells 5 b re , KA gen, 30 kDa) Induced > 2-fold
ANXA11 1 1.013 1.25 2.141 1.846 Annexin A11 CTDBPL 1 2.83 1.12 2.42
2.10 Carbcaryl-terminal despotin, RNA polymerase II, polypeptide A,
small phosphotace-like Charged < 2-fold CALR 1 0.71 0.843 1.482
1.528 Calroticolla CBARA1 1 0.665 0.773 0.493 0.721 CHD4 1 0.763
0.811 0.918 0.84 Chromopolmelin helicase DNA-binding protein 4
COLITA1 1 1.749 1.162 1.012 0.84 Collagen, type XVII, .sigma.1
CTDSP1 1 0.71 1.00 1.25 1.09 Cathoxyl-terminal domain, RNA
polymerase II, polypeptide A, small phophatase 1 CTDSP2 1 1.01 1.21
0.82 1.71 Cathoxyl-terminal domain, RNA polymerase II, polypeptide
A, small phophatase 2 21 1 1.842 0.861 0.722 0.05 Contains the 3'
end of a normal gene similar to NY.REPLY antigen DLAT 1 1.54 0.828
1.001 0.761 Dihydroll dly Soxotyltransferase ( ) component of
pyruvain dehydrogenesis complex EPPK1 1 0.783 0.880 1.718 0.859
Epiplakin I FBL 1 1.54 1.12 1.09 0.89 Creolin kinase II &
subunit complete cds, G22F1 1 1.15 1.183 1.18 0.792 Thyroid
autoantigen, 70 kDa (Ku antigen) GMEB3 1 0.084 0.782 0.006 0.745
Glucoso modulotory element-binding protein 2 GOLGA5 1 1.008 0.028
0.731 1.494 Golgi autoantigen, golgin subfamily a .delta. GOLGB1 1
0.838 0.701 0.56 0.858 Golgi autoantigen, golgin subfamily b,
macrogolgin with transmembrain 1 HARS 1 1.27 1.12 0.83 0.33
Hindlydl-IRNA synthetase HARSL 1 1.12 1.06 0.85 1.00 Hindlydl-IRNA
synthetase-like IMP-2 1 1.016 1.280 0.026 0.003 Insulin growth
mRNA, mRNA binding protein 2 LAD1 1 0.788 0.081 0.254 0.863 Elusion
(LAD) gene, complete cds LMD4 1 0.0 1.190 1.183 0.859 LIM domain
only 1 NB 1 0.004 0.08 0.180 0.814 Nadeoatenta PDHB 1 1.070 1.11
0.745 0.818 dehydrogen PDX1 1 1.103 0.785 0.046 0.520 R3-binding
protein PMNC1B 1 0.058 0.888 1.558 0.733 Polymyosit autoantigen 2,
110 kDa POLR1B 1 0.07 1.00 0.58 1.03 Polymerase (RNA) I polypeptide
B, 128 kDa POLR1B 1 0.00 0.06 1.01 1.31 Hypothetical protein
B100P880 POLR2B 1 1.00 1.02 0.09 0.74 Polymerase (RNA) II
(DNA-directed) polypeptide B, 110 kDa POLR2C 1 1.32 1.00 1.52 1.88
Polymerase (RNA) II (DNA-directed) polypeptide C, 33 kDa POLR2F 1
0.03 1.00 1.17 0.80 Polymerase (RNA) II (DNA-directed) polypeptide
F POLR2G 1 1.03 1.02 0.74 0.89 Polymerase (RNA) II (DNA-directed)
polypeptide G POLR2H 1 0.85 0.88 1.03 1.15 Polymerase (RNA) II
(DNA-directed) polypeptide H RALY 1 1.558 0.780 1.453 0.612
RNA-binding protein genic, human RMP-associated with lethal
swallows SART1 1 1.930 1.188 1.102 0.88 Squamous cell antigen
recogniaed by T cells SL 1 1.022 0.380 1.059 1.111 H. sapiens,
similar to e e-like antigen, clone, 3000 IMAGE:3139311, mRNA,
complete cds. SNRPD1 1 0.704 0.678 0.012 0.817 Small nuclear
ribonucleoprotein D1 polypeptide, 10 kDa SNRPG 1 1.28 1.03 1.28
1.12 Small nuclear ribonucleoprotein polypeptide G SOX12 1 1.804
0.771 1.24 1.882 SRY-box B SP110 1 1.149 1.028 0.7 0.021 SP110
nuclear body protein SPTBN 1 0.81 0.08 0.88 0.78 Spectelm, 8, non
oytis 1 SSA1 1 0.738 0.019 0.087 0.08 antigen A2191 kDa,
ribonucleoprotein autoantigen SSA101) SSA2 1 0.008 0.037 0.871
1.584 H. sapiens cDNA FL113838 , clone 17 , highly similar to human
80 kDa ribonucleoprotein RNA SSA3 1 0.598 0.88 0.83 0.81 H. sapiens
cDNA FL113838 , clone 17 , highly similar to human 80 kDa
ribonucleoprotein RNA SSMA1 1 1.254 1.17 1.038 0.815 nuclear
autoantigen 1 SSSMA1 1 1.837 1.694 1.035 1.283 autoantigen 1 XRCC8
1 1.87 1.141 0.883 0.725 X-ray repair complementing defensive
repels in Chilo cells & basals rejoining; Ku autoantigen,
80kDa) indicates data missing or illegible when filed
Sequence CWU 1
1
6118DNAartificial sequenceprimer 1tcccatcacc atcttcca
18221DNAartificial sequenceprimer 2catcacgcca cacgagtttc c
21322DNAartificial sequenceprimer 3gaactgctgc aggaggtgat aa
22420DNAartificial sequenceprimer 4ggcacattca gagaaggagt
20527DNAartificial sequenceprimer 5ccaaaatctg tcatcaaatt gagtatt
27623DNAartificial sequenceprimer 6ccagccttca tccagtttta tct 23
* * * * *